fphys-11-00855 July 16, 2020 Time: 11:16 # 1

REVIEWpublished: 16 July 2020

doi: 10.3389/fphys.2020.00855

Edited by:Alexei Tepikin,

University of Liverpool,United Kingdom

Reviewed by:Kazi Mirajul Hoque,

University of Maryland School ofMedicine, Baltimore, United States

Michael Chvanov,University of Liverpool,

United Kingdom

*Correspondence:Viktória Venglovecz

venglovecz.viktoria@med.u-szeged.hu

†These authors have contributedequally to this work

Specialty section:This article was submitted to

Gastrointestinal Sciences,a section of the journalFrontiers in Physiology

Received: 11 May 2020Accepted: 25 June 2020Published: 16 July 2020

Citation:Becskeházi E, Korsós MM,

Eross B, Hegyi P and Venglovecz V(2020) OEsophageal Ion Transport

Mechanisms and Significance UnderPathological Conditions.

Front. Physiol. 11:855.doi: 10.3389/fphys.2020.00855

OEsophageal Ion TransportMechanisms and Significance UnderPathological ConditionsEszter Becskeházi1†, Marietta Margaréta Korsós1†, Bálint Eross2, Péter Hegyi2,3,4 andViktória Venglovecz1*

1 Department of Pharmacology and Pharmacotherapy, University of Szeged, Szeged, Hungary, 2 Institute for TranslationalMedicine, Szentágothai Research Centre, Medical School, University of Pécs, Pécs, Hungary, 3 Division of Gastroenterology,First Department of Medicine, Medical School, University of Pécs, Pécs, Hungary, 4 First Department of Medicine, Universityof Szeged, Szeged, Hungary

Ion transporters play an important role in several physiological functions, such as cellvolume regulation, pH homeostasis and secretion. In the oesophagus, ion transportproteins are part of the epithelial resistance, a mechanism which protects theoesophagus against reflux-induced damage. A change in the function or expressionof ion transporters has significance in the development or neoplastic progressionof Barrett’s oesophagus (BO). In this review, we discuss the physiological andpathophysiological roles of ion transporters in the oesophagus, highlighting transportproteins which serve as therapeutic targets or prognostic markers in eosinophilicoesophagitis, BO and esophageal cancer. We believe that this review highlightsimportant relationships which might contribute to a better understanding of thepathomechanisms of esophageal diseases.

Keywords: ion transport, oesophagus, Barrett oesophagus, eosinophilic oesophagus, oesophageal cancer

INTRODUCTION

Ion and water transport play a crucial role in the development of gastrointestinal (GI) diseases, suchas cystic fibrosis or diarrhea. Therefore, a number of studies have been conducted to identify thepresence and function of transport proteins on GI cells, especially in exocrine glands, the stomachand the colon (Toth-Molnar et al., 2007; Venglovecz et al., 2008, 2011; Farkas et al., 2011; Hegyiet al., 2011; Czepan et al., 2012; Judak et al., 2014) both under physiological and pathophysiologicalconditions. In contrast, the literature on ion transport in the oesophagus is limited. The oesophagealepithelium (OE) is built up from squamous epithelial cells (SECs) arranged in stratified layers.Although the OE is not a typical secretory epithelium, its ion transport processes are of greatimportance with regard to epithelial resistance (Orlando, 1986; Goldstein et al., 1994; Gunther et al.,2014). OE resistance is an important defense mechanism which prevents cells from reflux-inducedacidosis (Fujiwara et al., 2005). OE resistance involves a number of factors, such as ion transportprocesses through apical and basolateral membranes. Disturbances of oesophageal ion transportmight play a role in certain pathological conditions, such as eosinophilic oesophagitis (EoO),Barrett’s oesophagus (BO) and oesophageal cancer (OC). This review summarizes our knowledge ofthe expression and function of oesophageal ion transporters and elucidates their role in oesophagealdiseases. We believe that this review highlights important relationships which might contribute toa better understanding of the pathomechanisms of oesophageal diseases.

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STRUCTURE OF THE OESOPHAGUS

The oesophagus consists of four layers, namely the mucosa,submucosa, muscularis externa, and adventitia. Epithelial cellsare located in the mucosa and submucosa layers. The mucosalayer is composed of squamous epithelial cells arranged in severalrows. There are differences in the thickness and keratinizationof the mucosa between species (Figure 1). In the oesophagusof rodents, squamous cells are arranged to 4 to 5 layers and aretypically keratinised. In humans and pig’s oesophagus, squamouscells are located in several layers, up to 10–15, and the cells are notkeratinised. The submucosa layer contains connective tissue cells,blood and lymphatic vessels, nerves, and glands. Interestingly,there are no glands in the oesophagus of rodents, while theyoccur in large numbers in the oesophagus of humans and pigs.The submucosal glands (SMGs) are tubuloacinar glands thatoccur scattered throughout the submucosa. SMGs secrete mainlymucus, HCO3

− and growth factors, and their primary function isto lubricate the oesophagus and to protect it from the damagingeffects of the refluxed acid. The submucosa layer is followed bythe muscular externa that is composed of smooth and striatedmuscle, while adventitia is mostly composed of connective andadipose tissue. Ion mechanisms on SECs have been primarilystudied in rabbit, whereas pigs proved to be a suitable animalmodel for studying ion transport processes associated with SMGs.The next section describes the ion transporters so far identifiedon SECs and SMGs.

PRESENCE AND ROLE OF IONTRANSPORTERS UNDERPHYSIOLOGICAL CONDITIONS

Cl− ChannelsChloride (Cl−) transport is essentially a two-step processthat involves the entry of Cl− into the cell across thebasolateral membrane and its secretion into the lumen throughthe apical membrane (Frizzell and Hanrahan, 2012). Theseprocesses involve various transport proteins, of which Cl−is usually taken up by cells through the Na+/K+/2Cl− co-transporter (NKCC) and released to the lumen through thecystic fibrosis transmembrane conductance regulator (CFTR).Epithelial Cl− transport provides an electrical driving forcefor the paracellular transport of Na+ from the basolateralside to the lumen. As a result, the osmotic pressure in thelumen increases, which promotes the movement of water intothe lumen (Frizzell and Hanrahan, 2012). Oesophageal Cl−transport in the squamous epithelium and SMGs was mainlyinvestigated by Abdulnour-Nakhoul et al. (2002) (Tables 1, 2).Using isolated rabbit OE, they showed the presence of Cl−conductance at both the basolateral and the apical membrane ofoesophageal epithelial cells (OECs) (Abdulnour-Nakhoul et al.,2002). The basolateral Cl− conductance was Ca2+-dependentand flufenamate-sensitive. However, the CFTR activator, cyclicadenosine monophosphate (cAMP), or the anion exchanger(AE) inhibitor, 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid

(DIDS), did not affect Cl− conductance. In addition, Cl−transport was independent of the presence of extracellularHCO3

−, indicating that basolateral Cl− conductance is believedto be a Ca2+-activated Cl− channel (CaCC). In contrast, apicalCl− conductance played only a minor role in Cl− transportindicating that there is no significant transcellular Cl− transportacross squamous cells. Transcellular Cl− transport has greaterimportance in SMGs, where fluid secretion is more significant.Apical Cl− transport in SMGs can be linked to the CFTRCl− channel (Abdulnour-Nakhoul et al., 2011). Using differentapproaches, the CFTR has been detected in the acinar cells andat the apical membrane of intra- and interlobular ductal cells ofpig oesophageal SMGs. In addition, using different activators andinhibitors it has been demonstrated that the CFTR Cl− channelis functionally active and is expected to be involved in HCO3

−

secretion in two ways: the CFTR (i) changes its permeabilityand secretes HCO3

− directly into the lumen and (ii) providesextracellular Cl− for the function of the apical Cl−/HCO3

−

exchanger, similarly, to the pancreas or the salivary gland (Leeet al., 2012). The transcellular transport of Cl− is mediated by thebasolateral AE2, through which Cl− enters the cell.

K+ ChannelsPotassium (K+) channels have an extensive family, whichincludes voltage-dependent K+ channels, Ca2+-activated K+channels, or ATP-regulated K+ channels (Heitzmann and Warth,2008). Epithelial K+ channels can be found on both thebasolateral and luminal membranes of the epithelial cells wherethey serve different functions. Voltage-dependent K+ channelsare usually expressed on the basolateral membrane, where theydetermine the membrane potential of the cell, provide anelectrochemical driving force for the transport of other ions,such as Na+ or Cl− and also play a role in the recycling of K+or the regulation of cell volume (Cotton, 2000). K+ channelson the apical membrane mediate the secretion of K+ into thelumen and therefore determine the K+ concentration of thesecreted fluid. Using chamber studies have shown the presenceof K+ conductance at the basolateral membrane of rabbit OECs(Goldstein et al., 1993; Khalbuss et al., 1993). The K+ channelis active under basal conditions and regulates transepithelialNa+ uptake and cell volume after osmotic stress. The basolateralK+ channel has also been shown to be important in regulatoryvolume decrease (RVD) in isolated rabbit oesophageal cells andhuman OECs (Snow et al., 1993; Orlando et al., 2002). RVD playsrole in the restitution of cell volume after an osmotic swelling. Inmost cases, RVD is mediated through the efflux of K+ and Cl−through the basolateral membrane, followed by water transport.Snow et al. (1993) confirmed the significance of K+ efflux inRVD and also showed that acidic pH inhibits K+ channel activity,indicating that reduced RVD is probably involved in the reflux-induced oesophageal injury. In addition, using pharmacologicalinhibitors (e.g., DIDS and diphenylamine-2-carboxylate) orintracellular Cl− removal, the authors showed that RVD alsodepends on Cl− efflux (Snow et al., 1993). Orlando et al. (2002)made similar observations in human oesophageal cells, with thedifference that they also identified a later, second mechanism

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FIGURE 1 | Histological comparison of rodent and human/pig oesophagus. The most common difference between rodent and human/pig oesophagus is that inrodents the mucosa layer is thinner and the lack of submucosal glands. SMG, submucosal gland.

underlying RVD regulation: using R(+)-butylindazone, a KCl co-transporter (KCC) inhibitor, they detected late RVD inhibition(Orlando et al., 2002). KCC is an electroneutral transporter thatmediates the coupled transport of 1 K+ and 1 Cl− for whichthe energy is provided by the K+ gradient maintained by theNa+/K+ ATPase pump (Haas and Forbush, 1998, 2000). TheKCC family has four isoforms (KCC1-4) which show high levelsof homology in their amino acid sequence. Although KCC1ubiquitously expressed, the presence of KCC2 and -3 mainlylimited to the nervous system. Abnormal regulation of KCC genesleads to the development of hematological diseases such as sicklecell anemia, which causes cellular dehydration in red blood cells(Rees et al., 2015). In the oesophagus, K+ and Cl− transportacross the basolateral membrane of SECs present a protectivemechanism by which cells try to maintain the cell volume againsthypo-osmotic shock under acidic pH which has a significance inreflux disease (Table 1).

Na+/K+/2Cl− Co-TransporterThe Na+/K+/2Cl− co-transporter (NKCC) mediates theelectroneutral uptake of 1 Na+, 1 K+ and 2 Cl− ions acrossthe plasma membrane (Russell, 2000). The NKCC has twoisoforms in humans (NKCC1 and NKCC2), which are encodedby two different genes (SLC12A1 and SLC12A2). The NKCC isprimarily located on the basolateral membrane of various cellsand plays a role in several physiological functions, such as fluidsecretion in exocrine glands (Haas and Forbush, 2000) smooth

muscle cell contraction (Akar et al., 2001) and early neuronaldevelopment (Ben-Ari et al., 2007). In most cells, NKCC interactswith KCC. The effect of the two transporters is opposite. KCCmediates Cl− efflux, whereas NKCC promotes the uptake of Cl−.The coordinated operation of the two transporters ensures theproper Cl− concentration in the cell. NKCC activity is greatlyinfluenced by intracellular Cl− concentration, which acts as anegative feedback mechanism (Rocha-Gonzalez et al., 2008).The mechanism by which cytoplasmic Cl− regulates NKCC isnot fully understood, but probably Cl− is able to modify thephosphorylated state of the cotransporter. Loss of functionmutations of NKCC2 leads to the development of type 1 Bartter’ssyndrome, in which the reabsorption of salt is reduced in thethick ascending limb of Henle’s loop (Castrop and Schiessl,2014). In oesophageal SECs, the NKCC is primarily involvedin cell volume regulation (Tobey et al., 1995). Functionalstudies on rabbit OE have shown that bumetanide-sensitiveNa+/K+/Cl− transport is activated by acidic pH, is linked tothe NKCC and induces cell oedema by ion uptake (Tobey et al.,1995). K+ and Cl− conductances work in opposite directionsand promote cell shrinkage. NKCC1 is present in the humanoesophagus, in the lower and middle layers of squamous cells(Shiozaki et al., 2014a). The NKCC is also present in SMGs,where it plays an essential role in fluid secretion (Abdulnour-Nakhoul et al., 2011). Immunostaining of pig oesophagusshows strong, positive staining against NKCC1 antibody onthe basolateral membrane of mucous cells and interlobular

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TABLE 1 | Expression of ion transporters in oesophageal squamous epithelial cells (SECs).

Ion transporters in oesophageal squamous epithelial cells (SECs)

Family Type or isoform Physiological role Localization Species References

Cl− channel CaCC Regulates intracellular Cl−

concentrationBasolateral Rabbit Abdulnour-Nakhoul et al., 2002

K+ channel N/A Regulates transepithelial Na+ uptakeand cell volume and also modulatesRVD by K+ efflux

Basolateral Rabbit Goldstein et al., 1993; Khalbuss et al.,1993; Snow et al., 1993

Human Orlando et al., 2002

KCC N/A Secondary regulatory mechanism ofRVD

Basolateral Human Orlando et al., 2002

NKCC NKCC1 Operates at acidic pH and regulatescell volume

N/A Rabbit Tobey et al., 1995

NHE NHE1 Causes cellular alkalisation by extrudingH+ out of the cells

Basolateral Rabbit Powell et al., 1975; Layden et al., 1990,1992; Abdulnour-Nakhoul et al., 1999;Shallat et al., 1995

Rat Shallat et al., 1995

Human Goldman et al., 2010; Guan et al.,2014; Laczko et al., 2016; Ariyoshiet al., 2017

AE Na+-dependent Causes cell alkalisation, mediates Na+,Cl− and HCO3

− transport through theplasma membrane

Basolateral Rabbit Bonar and Casey, 2008; Tobey et al.,1993

Na+-independent Causes cell acidification, mediates Cl−

and HCO3− transport through the

plasma membrane

non-selective cation channel ENaC (?) Equally permeable for monovalentcations (Li+, Na+ and K+), the exactrole is not known

Apical Rabbit Awayda et al., 2004

AE, anion exchanger; CaCC, Ca2+-activated Cl− channel; KCC, KCl co-transporter; NKCC, Na+/K+/2Cl− co-transporter; NHE, Na+/H+ exchanger; RVD, regulatoryvolume decrease; N/A, not available.

TABLE 2 | Expression of ion transporters in oesophageal submucosal glands (SMGs).

Ion transporters in oesophageal submucosal glands (SMGs)

Family Type or isoform Physiological role Localization Species References

Cl− channel CFTR Contributes to luminal HCO3− secretion Apical Pig Abdulnour-Nakhoul et al., 2011

NKCC NKCC1 Regulates cell volume and provides Cl− for HCO3− secretion Basolateral

AE AE2 Facilitates the influx of Cl− and the efflux of HCO3− Basolateral

SLC26A6 Mediates HCO3− transport into the lumen Apical

NBC NBC1 Contributes to luminal HCO3− transport Basolateral Abdulnour-Nakhoul et al., 2005, 2011

AE, anion exchanger; CFTR, cystic fibrosis transmembrane conductance regulator; NBC, Na+/HCO3− co-transporter; NKCC, Na+/K+/2Cl− co-transporter.

ducts. Bumetanide, a specific NKCC1 inhibitor, inhibits HCO3−

secretion, indicating that NKCC1 is involved in the process,presumably by providing the Cl− necessary for HCO3

− secretion(Abdulnour-Nakhoul et al., 2011).

Na+/H+ ExchangersNa+/H+ exchangers (NHEs) are electroneutral exchangers with astoichiometry of 1 Na+:1 H+. For the transport of Na+ and H+,NHEs use the energy that is provided by the electrochemical Na+gradient maintained by the Na+/K+-ATPase. The N-terminalmembrane domain of the exchanger is responsible for thetransport of ions, whereas NHE is regulated through theC-terminal cytoplasmic domain (Slepkov et al., 2007). NHEs

are involved in a wide range of biological functions, such ascell proliferation (Grinstein et al., 1989) cell migration (Denkeret al., 2000) and cell volume regulation (Demaurex and Grinstein,1994). One of their most important roles is cell alkalisationvia mediating the exchange of intracellular H+ for extracellularNa+. Excess Na+ is removed from the cell by the Na+/K+-ATPase and Na+/Ca2+ exchanger (Slepkov et al., 2007). NHEsare encoded by the SLC9A gene family, which includes ninemembers (NHE1–NHE9). NHE1 is ubiquitously expressed, whilethe other isoforms are restricted to a specific tissue. Abnormalfunction of NHEs is associated with a number of neurologicaland cardiac diseases (Donowitz et al., 2013). Most NHEs areactivated by acidification (Lacroix et al., 2004) and can be blocked

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by amiloride (Kleyman and Cragoe, 1988). Early micro-electrodestudies on rabbit oesophagus have described an amiloride-sensitive short-circuit current at the serosal and mucosal sidesof the oesophagus (Powell et al., 1975). Subsequently, thepresence of a Na+/H+ exchange mechanism was proved byLayden et al. (1990). They identified a Na+-dependent, alkalizingtransport system in rabbit oesophageal cells which was sensitiveto amiloride and was activated at physiological pH ranges(Layden et al., 1990). Further functional studies have confirmedthe presence of NHEs in rabbits (Layden et al., 1992; Abdulnour-Nakhoul et al., 1999) and humans (Tobey et al., 1998) where pHiregeneration depends on extracellular Na+ and occurs throughthe basolateral membrane (Abdulnour-Nakhoul et al., 1999).Reverse transcription-polymerase chain reaction (RT-PCR) andimmunoblot analysis confirm the presence of NHE1 in rat andrabbit oesophageal cells (Shallat et al., 1995). Taken together thesedata suggest that NHE1 is present on the basolateral membrane ofrodent oesophageal cells, where it is likely to regulate the pHi andprotect cells against reflux-induced acidity by extruding H+ outof the cells. In contrast, results on NHE1 expression and functionin humans are somewhat contradictory. Immunohistochemical(IHC) studies on human oesophageal tissue specimens haveshown the absence or weak expression of NHE1 in normalsquamous mucosa (Goldman et al., 2010; Guan et al., 2014;Laczko et al., 2016; Ariyoshi et al., 2017), while functional assaysperformed on the human oesophageal cell line and primarycultures detect a high degree of NHE activity (Tobey et al., 1998;Fujiwara et al., 2005). Even if NHE is present on OECs undernormal conditions, the role of NHE in the oesophagus is muchmore important under pathological conditions as detailed below.

Cl−/HCO3− Exchangers and Na+/HCO3

−

Co-TransportersCl−/HCO3

− exchangers or AEs mediate Cl− and HCO3−

transport through the plasma membrane (Bonar and Casey,2008). AEs either alkalise or acidify cells, depending on thedirection of HCO3

− transport. The AE family comprises a largenumber of transporters, which are encoded by two gene families(SLC4A and SLC26A) (Bonar and Casey, 2008). The SLC4Afamily comprises three transporter types: Na+/HCO3

− co-transporters (NBCs), Na+-dependent Cl−/HCO3

− exchangers(NDCBE and NCBE) and Na+-independent Cl−/HCO3

−

exchangers (AE1, AE2, and AE3). Five members of the SLC26Afamily (SLC26A3, SLC26A4, SLC26A6, SLC26A7, and SLC26A9)differ most in their different Cl−: HCO3

− stoichiometry andtherefore in their electrogenicity. AE family members differ inmany aspects, such as tissue expression, cell surface localizationand physiological function. Abnormal expression of SLC26A2-4genes associated with rare, recessive diseases, such as diastrophicdysplasia, congenital chloride diarrhea or Pendred syndrome,respectively (Kere, 2006).

Two types of AE are found in oesophageal SECs (Tobeyet al., 1993): a Na+-dependent AE and a Na+-independentCl−/HCO3

− exchanger. Although both transporters areelectroneutral, their effects are opposite. The Na+-dependentAE plays an important role in cell alkalisation, along with the

NHE, while the Na+-independent AE acidifies cells, preventingover-alkalisation. Na+-independent AEs mediate the exchangeof 1 Cl− for 1 HCO3

−, while Na+-dependent AEs transportNa+, in addition to HCO3

− and Cl−. Both Na+-dependent andNa+-independent AEs localize to the basolateral membrane andconduct HCO3

− transport from or toward the blood (Laydenet al., 1992; Tobey et al., 1993). Oesophageal HCO3

− secretionis much more related to SMGs (Abdulnour-Nakhoul et al.,2005, 2011). A basal and carbachol-stimulated movement ofHCO3

− was detected which was inhibited by DIDS in perfusedpig oesophagus. IHC staining showed the presence of an NBCand AE2 on intra- and interlobular ducts and serous demilunes,indicating that HCO3

− secretion is probably due to these twotransporters. These transporters are localized to the basolateralmembrane of ductal cells, and they presumably provide HCO3

−

and Cl− for luminal HCO3− secretion (Abdulnour-Nakhoul

et al., 2011). In contrast, at the luminal membrane of interlobularducts, studies have shown the messenger RNA (mRNA) andprotein expression of Slc26A6, which mediate HCO3

− transportinto the lumen, in cooperation with the CFTR Cl− channel(Abdulnour-Nakhoul et al., 2011). Luminal HCO3

− secretionis believed to protect the ooesophagus by neutralizing theacidic refluxate.

Other Ion TransportersAwayda et al. (2004) identified non-selective cation conductanceat the apical membrane of rabbit OECs which is equallypermeable to monovalent cations, such as Li+, Na+, and K+(Awayda et al., 2004). In addition, western blotting showedthat this non-selective cation channel contains all three sub-groups of the epithelial Na channel (ENaC), in both nativerabbit OE and HET-1A cells, although it does not have thecharacteristics of epithelial Na+ channels and is not inhibitedby amiloride. The ENaC mediates cation uptake, although itsexact function is unknown. In addition, similarly, to otherepithelial cells, the Na+ gradient across the basolateral membraneis maintained by Na+/K+-ATPase whose presence has beendetected on both squamous cells (Orlando et al., 1981, 1984) andon the basolateral membrane of acini and ductal cells of SMGs(Abdulnour-Nakhoul et al., 2011).

Figure 2 shows the presence and localization of iontransporters on rabbit SECs (Figure 2A) and pig SMGs(Figure 2B). In the case of SECs, transporters are mainlylocalized to the basolateral membrane, and their primary roleis to increase the resistance of these cells to acidic refluxby restoring changes in cell volume and intracellular pH. Incontrast, apical and basolateral ion transporters on SMGs areprimarily involved in HCO3

− secretion, which is secreted intothe lumen via the SLC26a6 AE on the apical membrane ofintra-interlobular ducts. The secreted HCO3

− neutralizes theacidic gastric contents thereby largely prevents the diffusion ofthe proton into the oesophageal tissue. In conclusion, underphysiological conditions, ion transport processes through theSECs and SMGs represent an important defense mechanismthat protects the lower layers against the harmful effect ofacidic GI contents. Inadequate or altered function of these

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FIGURE 2 | The presence and localization of ion transporters on squamous epithelial cells (SECs) and submucosal glands (SMGs). The schematic diagram shows arabbit SEC (A) and a ductal cell of pig SMG (B). AE, anion exchanger; CA, carbonic anhydrase; CaCC, Ca2+-activated Cl- channel; CFTR, cystic fibrosistransmembrane conductance regulator; KCC, KCl co-transporter; NBC, Na+/HCO3

- co-transporter; NHE, Na+/H+ exchanger; NKCC, Na+/K+/2Cl- co-transporter.

transporters plays a role in the development and/or progressionof oesophageal diseases, such as EoE, BO or OC.

ROLE OF ION TRANSPORTERS UNDERPATHOLOGICAL CONDITIONS

Eosinophilic OesophagitisEosinophilic oesophagitis is a chronic inflammatory diseaseinduced by food antigens (Furuta and Katzka, 2015). EoEis characterized by basal zone hyperplasia (BZH), dilatedintercellular spaces (DISs) and the filtration of eosinophilicgranulocytes into the oesophageal mucosa (Collins, 2008).Interleukin 13 (IL-13) plays a major role in EoE pathology.IL-13 is highly up-regulated in inflamed tissue and regulatesthe expression of several key molecules (Blanchard et al.,2010). Studies have reported altered expression of two iontransporters [NHE3 and anoctamin 1 (ANO1)] in EoE (Zenget al., 2018; Vanoni et al., 2020). The mRNA and proteinexpression of both NHE3 and ANO1 significantly increasesin biopsy samples obtained from EoE patients and in IL-13-treated OECs and model systems. Both NHE3 and ANO1 areup-regulated by the transcription factor signal transducer andactivator of transcription 6 (STAT6) and are positively correlatedwith disease progression (Zeng et al., 2018; Vanoni et al.,2020). Pharmacological NHE3 inhibition blocks IL-13-inducedDIS formation, indicating the involvement of NHE3 in DISformation (Zeng et al., 2018). NHE3 mediates the exchange ofintracellular H+ with extracellular Na+. Proton accumulation inthe intercellular space creates osmotic conditions which favorwater transport and, as a result, DIS formation (Zeng et al., 2018).

In contrast to NHE3, increased ANO1 expression is associatedwith BZH (Vanoni et al., 2020). ANO1 is an apical Ca2+-activated Cl− channel which is also permeable to HCO3–(Junget al., 2013). Application of ANO1 inhibitor or short hairpinRNA (shRNA) shows that this Cl− channel interacts with cellcycle regulatory proteins, such as TP63 and phosphorylatedcyclin-dependent kinase 2 (p-CDK2), thereby enhancing cellproliferation. In addition, Cl− efflux through ANO1 likely plays arole in intracellular Cl− depletion, which is a prerequisite for celldivision. These results indicate that both NHE3 and ANO1 arepart of the IL-13-mediated transcriptional cascade, which leadsto the histopathological features of EoE. Therefore, inhibition ofthese transporters could be a potential therapeutic target for EoEtreatment (Vanoni et al., 2020).

Gastro-Oesophageal Reflux Disease andBarrett’s OesophagusBarrett’s oesophagus is a condition in which the normalsquamous epithelium is replaced by columnar cells (Spechler andSouza, 2014). It is the result of long-term gastro-oesophagealreflux disease (GERD) and a type of adaptation to the alteredenvironment (Spechler and Souza, 2014). Many factors playa role in BO development, although the exact underlyingmechanism is unclear. Several studies have been conducted inwhich normal epithelial cells were exposed to acidic pH, bileacids or a combination of the two and molecular changes andcell survival were investigated. Lin et al. (2008) identified DIDS-and ethyl-isopropyl amiloride (EIPA)-sensitive and HCO3

−-dependent transport mechanisms in rat oesophageal tissue uponHCl administration, indicating the involvement of an NHE and

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one of the SLC26 AEs in the stimulatory effect of HCl (Linet al., 2008). Increased ionic movement due to acid exposureis likely a protective mechanism and might play a role in theGERD pathomechanism. Acidic pH decreases cell viability andincreases reactive oxygen species production in normal OECs, inwhich NHE1 plays a protective role (Park et al., 2015). Similarresults have been found in human oesophageal cell lines, inwhich the cytoprotective effect of epidermal growth factor againstacid-induced cell damage is mediated by NHE1 through theCa2+/calmodulin and protein kinase C (PKC) pathway (Fujiwaraet al., 2005). Increased NHE1 expression is found in oesophagitis,GERD and BO, although it is unclear whether increased NHE1levels are the consequence or the cause of these diseases(Siddique and Khan, 2003; Fujiwara et al., 2005; Goldman et al.,2010; Laczko et al., 2016). According to Siddique and Khan(2003) NHE1 is involved in GERD development, presumably byacidifying the extracellular space (Siddique and Khan, 2003). Theauthors also found that in patients taking H2 receptor blockers,the mRNA level of NHE1 is significantly lower compared tountreated or antacid-treated GERD patients, assuming a specificinteraction between NHE1 and the histamine receptor in GERD.In contrast, the increased NHE1 expression in BO is more likelypart of a defense or adaptive mechanism which protects cellsagainst acid reflux (Goldman et al., 2010). This hypothesis is alsosupported by another study in which bile treatment alone or incombination with acid increased NHE1 expression in metaplastic(CP-A) and dysplastic (CP-D) oesophageal cell lines (Laczkoet al., 2016). Interestingly, NHE activity decreased in CP-A asa result of treatment but increased in CP-D cells in a dose-dependent manner. The reason probably is that CP-D cells arein a more advanced state and are more frequently exposed toacid or biliary reflux under pathological conditions. In additionto NHE1, NHE2, NBC, and Slc26a6 expression also increased inintestinal and non-intestinal metaplasia, which probably furtherenhances cellular resistance against reflux-induced cell damage(Laczko et al., 2016). These results suggest that increased NHEexpression found in BO is essentially a defense mechanism bywhich metaplastic tissue tries to adapt to the altered environment.Inhibition of the transporter promotes tumorigenesis because ofa decrease in intracellular pH.

Transient receptor potential channels (TRP) is a multifamilyof integral membrane proteins that found on several tissuesand cell types in animals (Nilius and Owsianik, 2011). TRPchannels function as non-selective cation channels and arepermeable to Na+; Ca2+ and Mg2. The TRP family can besubdivided into seven subfamilies: TRPC (canonical), TRPV(vanilloid), TRPM (melastatin), TRPP (polycystin), TRPML(mucolipin), TRPA (ankyrin), and TRPN (NOMPC-like). Thesechannels mediate a wide range of physiological functions, such assensations of temperature, pain, tastes, vision, olfaction, hearingand intracellular ion homeostasis. Some TRP channels act asa thermo-; osmolarity- or voltage sensor, and some of themactivated by membrane stretch or cytoplasm volume changes.The activated TRP channels cause depolarisation of the cellmembranes, which is essential in the development of actionpotential in excitable cells (Nilius and Owsianik, 2011). Therole of the TRP channels in the non-excitable cells is mostly

to mediate the ion homeostasis of the cells (Yu et al., 2016).TRPV1 acts as molecular acid sensor in the oesophagus whichactivated by acidosis (pH level under 6) (Holzer, 2008). TRPV1is overexpressed in the oesophageal mucosa of patients withnon-erosive reflux disease (NERD) and erosive esophagitis andalso in animal models of NERD (Guarino et al., 2010; Silvaet al., 2018). Increased levels of TRPV1 was also observed instress-induced oesophageal inflammation (Wulamu et al., 2019).The role of TRPV1 in inflammation can be explained by thefact that activation of the channel promotes the secretion ofinflammatory mediators (platelet-activating factor (PAF), Il- 1β, Il-6) that initiates the inflammatory cascade in the mucosaof the oesophagus. Therefore, inhibition of TRPV1 could bebeneficial in inflammatory conditions. Recently, Zhang et al.have shown that menthol inhibits acid-induced inflammationby inhibiting TRPV1 expression and TRPV1-mediated Ca2+

influx in oesophageal epithelial cells (Zhang et al., 2020). TRPV4is also a non-selective cation channel activated by mechanical,thermal, and chemical stimuli. Acid-sensitive TRPV4 channelsare mainly expressed in the epithelial cells of the basal andintermediate layers of the oesophagus (Shikano et al., 2011). Uedaet al. showed the presence of TRPV4 in human oesophagealbiopsy samples and HET-1A cell line. They demonstrated thatactivation of TRPV4 increases intracellular Ca2+ level anddecreases Il-8 production that plays a role in different cellularfunctions, such as cell division or viability (Ueda et al., 2011). Thepresence of functionally active TRPV4 has also been shown inmouse oesophageal keratinocytes, where activation of the channelcaused ATP release which affects the sensory nerves and mightalso be related to the pathophysiology of NERD (Mihara et al.,2011; Suzuki et al., 2015).

Oesophageal CancerOesophageal cancer is a malignant neoplasm, and the sixth-leading cause of cancer-related death worldwide (Klingelhoferet al., 2019). The two most common types of OC areoesophageal squamous cell carcinoma (ESCC) and oesophagealadenocarcinoma (EAC) (Enzinger and Mayer, 2003). Currenttreatment of OC includes surgery, chemo- or radiotherapy orthe combination of the three. Although OC treatment anddiagnosis have advanced, the 5-year survival rate is between15 and 20% and recurrence is frequent (Shapiro et al., 2015).Therefore, there is an urgent need to develop new therapiesthat can complement existing treatments or allow individualtherapies. Disturbances of ion transport processes are consideredto be of great importance in cancer research (Pedersen andStock, 2013; Djamgoz et al., 2014; Oosterwijk and Gillies, 2014;Stock and Schwab, 2015; Martial, 2016). Ion transporters arenot only involved in tumorigenesis by altering the membranepermeability of certain ions, but some transporters interact withother signaling molecules (kinases, transcription factors) thatinfluence the cell cycle or the metastatic potential of the cells.In recent years, several transporters and ion channels have beenidentified whose expression is altered in OC. In the following,those ion transporters are discussed, that might provide a targetpoint for novel treatment or serve as prognostic markers forESCC or EAC (Supplementary Table S1).

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K+ ChannelsDing et al. (2008a,b) identified two voltage-gated K+ channels,Eag1 (KV10.1, KCNH1) and hERG1 (KV11.1, KCNH2), whichare aberrantly expressed in ESCC and are correlated with poorprognosis (Ding et al., 2008a,b). Human ether a-go-go-relatedgene 1 (hERG1) over-expression has been found in BO and evenmore in dysplasia and adenocarcinoma, indicating that hERG1expression increases during BO progression to EAC (Lastraioliet al., 2006). To support this observation, Lastraioli et al. (2016)conducted a comprehensive study on hERG1 expression inboth mice and humans. The authors found increased hERG1expression in two different BO mice models. They also foundthat surgically induced BO more frequently develops in micewith hERG1 over-expression. The significance of hERG1 in BOprogression to EAC has also been demonstrated in human biopsysamples obtained from BO patients. Using monoclonal antibodyagainst hERG1, the 10-year follow-up showed that BO patientswith increased hERG1 expression have a higher risk of EACdevelopment, indicating that hERG1 can be used as a biomarkerto predict disease progression (Lastraioli et al., 2016).

Inhibitors and Therapeutic PerspectivesPotassium channel blockers are most commonly used asantiarrhythmic agents, however, research in recent years hasshown that there is a growing need for K+ channel inhibitorsin cancer therapy as well. Currently, AZD9291 is used inthe treatment of non-small cell lung cancer (Janne et al.,2015). AZD9291 is an epidermal growth factor receptor (EGFR)tyrosine kinase inhibitor (TKI) that is able to inhibit hERGchannels. Another EGFR TKI, gefitinib has also been provedto be effective in non-small cell lung cancer patients and hasshown to be beneficial in breast cancer as well (Garcia-Quirozet al., 2019). Experimental data demonstrated that gefitinibdose-dependently inhibited the proliferation of cancer cells andcombined administration of this drug with the antihistamine,astemizole resulted in a synergistic effect (Garcia-Quiroz et al.,2019). Although the two EGFR TKIs inhibit different channels,AZD9291 is specific for hERG channel, while gefitinib for Eag1,they have similar mechanisms of action, both of them prevent thechannel phosphorylation which is required for the activation ofthe channels (Zhang et al., 2008; Wu et al., 2012).

ANO1Shang et al. (2016) reported a correlation between anoctamin-1 (ANO1) expression and primary oesophageal tumor in twoindependent cohorts. The authors detected strong ANO1 stainingin the tumor region of surgical specimens obtained fromESCC patients, while the adjacent normal tissue and endoscopicbiopsies from normal mucosa and chronic oesophagitis werecompletely negative for this Cl− channel (Shang et al., 2016).In addition, ANO1 expression is correlated with the malignantprogression of precancerous oesophageal lesions, indicating thatANO1 can be used as a biomarker to predict disease progression.Shi et al. (2009) identified genes whose expression is altered inESCC and dysplasia. They found increased ANO1 levels at bothmRNA and protein levels in ESCC and dysplastic samples. Inaddition, they found a correlation between ANO1 expression

and tumor progression (Shi et al., 2004). Interestingly, in themRNA expression of ANO1, the authors found no significantdifference between normal mucosa and oesophagitis (Shi et al.,2009) although Vanoni et al. (2020) have shown increasedANO1 expression in EoE. Genetic inhibition of ANO1 decreasesproliferation in ESCC cell lines, confirming the role of ANO1in cell division. The mechanism underlying ANO1-inducedpromotion of cell proliferation is unclear, although, in headand neck squamous cell carcinoma, activation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulatedkinase (ERK) signaling pathway is involved in the proliferativeeffect of ANO1 (Duvvuri et al., 2012).

Inhibitors and Therapeutic PerspectivesSeveral inhibitors are known that can inhibit ANO1 with greateror lesser potency or selectivity, such as the small moleculeinhibitor, Ani9 (Seo et al., 2016) the benzofuran derivative,benzbromarone (Huang et al., 2012), the flavonoid, luteolin (Seoet al., 2017) the aminophenylthiazole, [T16A(inh)-A01] or thearylaminothiophene, CaCC(inh)-A01 (Namkung et al., 2011).Among them, luteolin has been shown as potential anticancertherapeutic agents for the treatment of prostate cancer (Seo et al.,2017). This inhibitor not only blocks ANO1 activity but alsodecreases its protein expression. Using PC-3, prostate cancercell line, administration of luteolin reduced the proliferationand migration of the cells (Seo et al., 2017). Similar resultswere observed with Ani9, T16A(inh)-A01 and CaCC(inh)-A01 inhibitors, which also reduced PC-3 cell proliferation andinduced apoptosis, by a TNF-α-mediated signaling pathway(Song et al., 2018). ANO1 inhibitors have also been shown to bebeneficial in ovarian cancer, where administration of LY294002reduced the growth of cancer cells by inhibiting the PI3K/Aktsignaling pathway (Liu et al., 2019). The mechanism of actionof ANO1 inhibitors is not yet known, and there is no evidenceto date that it has been used in the treatment of inflammatoryor cancerous diseases of the oesophagus, but it could be apromising target.

AEsShiozaki et al. (2017) characterized the role of AE1 and AE2in ESCC tumorigenesis. Analysis of ESCC samples showed thatthe AE1 staining intensity depends on the tumor length, whilethe focal or diffuse distribution of AE1 is correlated with thehistological type and pT stage (Shiozaki et al., 2017). With regardto 5-year survival, the staining score was not correlated withprognosis, although diffuse AE1 expression was associated withpoor survival in advanced stages (Shiozaki et al., 2017). AE1expression has been found in ESCC cell lines (TE5, TE8, TE9,and KYSE150), where it is involved in cell cycle regulationand the proliferation, apoptosis, migration and invasion ofESCC cells via the MAPK and Hedgehog signaling pathways(Shiozaki et al., 2017). In contrast to AE1, decreased AE2expression is associated with poor prognosis in ESCC patients(Shiozaki et al., 2018). There is a correlation between the AE2staining score and the pT stage. With regard to survival, lowAE2 expression predicts a worse prognosis. Interestingly, thesecorrelations were observed only in the invasive front of ESCC,

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not in the entire tumor. In contrast to AE1, AE2 expressionwas found in KYSE170, and TE13 ESCC cell lines and AE2knock-down decreased apoptosis and increased migration andinvasion of ESCC cells, indicating that AE2 regulates cell survivaland cellular movements. Studies have also demonstrated thatthe effect of AE2 is mediated by matrix metalloproteinases(MMPs) and their inhibitors. Altered AE2 expression is believedto affect tumorigenesis by altering intracellular pH. DecreasedAE2 expression favors intracellular alkalisation, which promotescancer cell metabolism (Persi et al., 2018).

Inhibitors and Therapeutic PerspectivesThe most commonly used inhibitors of anion transporters arethe stilbene derivatives, DIDS, its reduced form dihydroDIDS(H2DIDS) and 4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid (SITS). The negatively charged sulfonate groupof these inhibitors interact electrostatically with the anionbinding site of the transporter, resulting in a rapid, reversibleinhibition, followed by a slower covalent interaction betweenthe isothiocyanate group of the inhibitor and the free aminoacids of the AE. These inhibitors have low specificity and inhibitindividual isoforms with different potency (Humphreys et al.,1994; Alper et al., 1998). There are also differences in theefficacy of inhibitors between species and cell types. The oxonoldye, diBA(5)C4 has been shown to inhibit the AE1 isoformmore effectively than AE2, while polyaminosterols have higherselectivity to AE2 (Alper et al., 1998). Among the more recentlydiscovered AE inhibitors, 4,8-dimethyl-7-(m-bromobenzyloxy)-coumarin-3-acetic acid has been shown to be able to completelyand selectively inhibit the SLC26A3 exchanger, and the useof this agent showed a beneficial effect on constipation in amouse model (Lee et al., 2019) although, to our knowledge,there is currently no AE inhibitor which enters or underwent inclinical trials.

KCC3 and NKCC1Among KCC isoforms, KCC3 expression is associated with OCtumorigenesis (Shiozaki et al., 2014b). The presence of KCC3in the invasive front of ESCC is associated with a low survivalrate post-operatively. The mechanism underlying KCC3’s effecton tumor progression is unclear, but studies using ESCC celllines have shown that KCC3 is essential for the invasiveness ofcancer cells (Shiozaki et al., 2014b). NKCC1 is also a biomarker inESCC. NKCC1 expression is closely related to the differentiationof ESCC cells, and genetic or pharmacologic inhibition ofNKCC1 in ESCC cell lines significantly decreases the rate of cellproliferation, indicating that NKCC1 is involved in cell cycleregulation (Shiozaki et al., 2014a).

Inhibitors and Therapeutic PerspectivesThe loop diuretics, bumetanide and furosemide, are the mostcommonly used inhibitors of NKCCs. Both inhibitors are able toinhibit the co-transporter even at very low concentrations (0.5–5 µM). Diuretics have long been used to treat fluid retention,and in recent years their use has also emerged in the treatmentof certain neurological disorders. Therefore, several clinicalstudies have been conducted to test the effect of bumetanide in

schizophrenia, epilepsy or Parkinson’s disease (Ben-Ari, 2017).Although, the clinical use of this inhibitor is limited due toits ionized state at physiological pH and its high bindingto plasma proteins. Recent studies suggest that azosemide ismore potent than bumetanide and has better pharmacokineticproperties (Hampel et al., 2018). Bumetanide and furosemide athigher concentrations (100 µM–1 mM) inhibit KCCs as well.In addition, KCC also sensitive to DIDS, H2DIDS, and SITS,although the inhibitory effect of these stilbene derivatives highlydepends on external K+ (Delpire and Lauf, 1992). Currently,the most commonly used KCC inhibitor is dihydroindenyloxyalkanoic acid (DIOA), which selectively inhibits the transporter.The large-scale high-throughput screen on HEK293 cellsidentified more potent inhibitors, such as ML077 or VU0463271,(Delpire and Weaver, 2016) although none of these compoundsis currently under clinical development.

NHEAmong ion transporters, the expression of NHEs, especiallyNHE1, alters in a metaplasia-dysplasia-adenocarcinomasequence. Recent studies have demonstrated that NHE1 alsoplays a significant role in BO progression to EAC. Fitzgeraldet al. (1998) showed that NHE1 is involved in acid-induced cellproliferation in BO biopsy samples in a PKC-dependent manner(Fitzgerald et al., 1998). The cell proliferation-inducing effectof NHE1 is explained by increased NHE1 activity at acidic pH,which likely affects cell division by altering intracellular pH. Incontrast, bile acids alone or in combination with acid decreaseNHE1 activity in nitric oxide– and nitric oxide synthase–dependent manner in BO-derived CP-A cells, leading to cellularacidosis (Goldman et al., 2010). The decrease in intracellular pHfavors DNA damage, which increases the incidence of mutationsand, therefore, the risk of EAC progression (Goldman et al.,2010). However, this workgroup also showed that bile aciddamages lysosomes as well in JH-EsoAd1 and CP-A cells and theacidic content released from them increases NHE activity, whichforms an ionic environment that favors to apoptotic processes.Based on these findings, it is hypothesized that by the inhibitionof NHE, the cells avoid apoptosis, increasing the risk of cancertransformation (Goldman et al., 2011). The role of NHE1 inOC is contradictory. Guan et al. (2014) have shown that NHE1is over-expressed in EAC and inhibition of NHE1 expressionor blocking of NHE activity by amiloride decreases cancer cellproliferation and induces apoptosis in both EAC and ESCCcell lines and in nude mice xenografts. Moreover, amiloridein combination with guggulsterone (antagonist of frasenoidX receptor) proved to be even more effective than amiloridealone. These results indicate that contrast to metaplastic cells,inhibition of NHE does not enhance tumor progression but,on the contrary, inhibits it, suggesting that NHE behavesdifferently in cancerous and metaplastic cells. In contrast,Ariyoshi et al. (2017) found that NHE1 inhibition favors theproliferation, migration and invasion of ESCC cells because ofthe up-regulation of epithelial-mesenchymal transformation(EMT) markers and the inhibition of Notch signaling. The 5-yearoverall survival (OS) was significantly higher in ESCC patients

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with high NHE1 expression, indicating the possibility of usingNHE1 as a prognostic marker (Ariyoshi et al., 2017).

Inhibitors and Therapeutic PerspectivesThe most commonly used NHE1 inhibitors are amilorideand its derivatives such as EIPA (5-(N-ethyl-N-isopropyl)amiloride), DMA (5-(N,N-dimethyl) amiloride) and HMA(5-(N,N-hexamethylene) amiloride) that belongs to thepyrazinoylguanidine-type inhibitors, as well as the more specificcariporide and epripride that belongs to the benzoylguanidine-type inhibitors (Rolver et al., 2020). In the case of both types ofinhibitors, guanidine binds to the sodium binding site of theexchanger, thereby competes with the sodium for the bindingsite (Frelin et al., 1986). Numerous studies have suggested theuse of NHE inhibitors in the treatment of various types of cancer,such as breast or colon cancer (Miraglia et al., 2005; Rolveret al., 2020), however, clinical evaluation of NHE inhibitors iscurrently limited to cardiac disease, particularly in connectionwith ischemia-reperfusion injury, in which the cardioprotectiveeffect of cariporide and eniporide was demonstrated in twoclinical trials (Chaitman, 2003; Mentzer et al., 2008).

Ca2+ ChannelsOrai-1 is the pore-forming subunit of the calcium-releaseactivated channel. By interacting with the Ca2+ sensor stromalinteraction molecule 1 (STIM-1), Orai-1 mediates store-operatedCa2+ entry (SOCE). Orai-1 is involved in tumorigenesis in manycancer types, including OC (Vashisht et al., 2015). Zhu et al.(2014) reported increased Orai-1 expression in tumor tissuesobtained from ESCC patients. The Orai-1 expression is correlatedwith clinicopathological features, such as histological grade,T-stage classification and lymph node metastasis, and poor OSand recurrence-free survival rates. Increased Orai-1 expressionand intense oscillatory Ca2+ activities are also found in ESCCcell lines, which probably plays a role in carcinogenesis. The roleof Orai-1 in tumorigenesis has been confirmed using cell-basedassays in which Orai-1 knock-down decreased the proliferation,migration and invasion of ESCC cells and tumor growth in nudemice xenografts (Zhu et al., 2014). The proliferative effect ofOrai-1 might be inhibited by zinc, which selectively interactswith the histidine residue of Orai-1 and inhibits SOCE and Ca2+

oscillations in ESCC cells (Choi et al., 2018). RP4010, a specificOrai-1 inhibitor, also inhibits tumor growth via an unknownmechanism and is a potential therapeutic or adjuvant drug forESCC treatment (Cui et al., 2018). The role of voltage-gated Ca2+

channels has also been raised in relation to OC. Voltage-gatedcalcium channels are comprised of heteromultimeric complexesthat involve α1 protein in combination with various auxiliarysubunits, β, α2δ, and γ protein (Walker and De Waard, 1998).Four α2δ subunit genes have been identified (CACNA2D1-4) (Qin et al., 2002). The α2δ-3 subunit (CACNA2D3) isresponsible for Ca2+ influx, and its tumor suppressor functionwas also characterized. It has been shown, that CACNA2D3induces apoptosis, reduces cell motility, and provokes cellcycle arrest in G1 phase through transactivating p53/p21signaling pathways (Lipskaia and Lompre, 2004;Li et al., 2013).The potential tumor suppressor role of CACNA2D3 in the

pathomechanism of malignancies has been mentioned in severalstudies. Considering the GI tract, the promoter methylation ofthis gene is in association with poor prognosis of gastric cancer(Wanajo et al., 2008). Li et al. showed that downregulation ofCACNA2D3 could be detected in ESCC patients that showeda positive correlation with a poor progression and low overallsurvival rate (Li et al., 2013). A recent study indicates, thatoverexpression of CACNA2D3 can restore chemosensitivity ofESCC cells to cisplatin via inhibition of PI3K/Akt pathway,which is a promising finding regarding the therapy of ESCC(Nie et al., 2019).

TRPC6 proteins are non-selective cation channels, whichare permeable to cations (Ca2+, Na +, K+, Cs+ and Ba2+)iron, zinc and N-methyl-D-glucamine (Bouron et al., 2016).TRPC6 seems to be ubiquitous in the human body andinvolved in the regulation of intracellular Ca2+ concentration(Hofmann et al., 2000). There are several activators of the TRPC6channels, such as extracellular Ca2+ concentration,(Shi et al.,2004) diacylglycerol (DAG) and its analogs, such as 1-oleoyl-2-acetyl-sn-glycerol, and arachidonic acids (Aires et al., 2007)and hyperforin (Leuner et al., 2007). Literature data indicatethat TRPC6 is associated with tumorigenesis, tumor growth andelevated TRPC6 expression was detected in many cancer types(Santoni and Farfariello, 2011). It has been reported that TRPC6expression significantly higher in ESCC patients, and it correlateswith poor progression (Shi et al., 2009; Zhang et al., 2013). Shiet al. have shown, that inhibition of TRPC6 resulted in cell cyclearrest at G2 phase, reduced cell growth in ESCC cells and theformation of tumor in nude mice (Shi et al., 2009). Evaluatingthis finding (Ding et al., 2010) have raised the possibility thatinhibition of TRPC6 can increase the radiosensitivity of tumorcells and also inhibits angiogenesis in the therapy of ESCC.Similar results have been found by Zhang et al. Using an ESCCcell line, Eca109 they showed that administration of the TRPCinhibitor, SKF96365 arrested the cell cycle at G2/M phase, andin combination with radiation it significantly reduced in vitro cellproliferation and the tumor size in nude mice (Zhang et al., 2017).

The role of TRPVs has arisen primarily in the inflammatorydiseases of the oesophagus (GERD, NERD); although Huanget al. have also shown its importance in ESCC (Huang et al.,2019). They found that both TRPV1 and -4 are up-regulated inESCC, and overactivation of these channels can modulate theproliferation and migration of ESCC cells, by the regulation ofintracellular Ca2+ concentration (Huang et al., 2019).

Inhibitors and Therapeutic PerspectivesCa2+ channels are aberrantly regulated in most of the cancercells; therefore, great efforts are made to develop novel drugsthat are able to modulate the function of these channels.Inhibition of Ca2+ channels is a promising target in cancertherapy, and there are Ca2+ channel blockers that have alreadyundergone clinical trials or are under clinical development.The specific Orai-1 inhibitor, RP4010 completed a Phase I/Ibclinical trial in which the safety and efficacy of RP4010were evaluated in lymphoma. In addition, RP4010 significantlyreduced cell division, especially in combination with anticanceragents in pancreatic ductal adenocarcinoma (Khan et al., 2020).

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The recently identified SOR-C13 is an inhibitor of TRPV6channels that selectively binds to the channel and preventsthe influx of Ca2+ into the cells. SOR-C13 has been shownto be effective in ovarian cancer (Xue et al., 2018) and iscurrently undergoing Phase I clinical trials to determine thebest dose and side effect profile of this drug, in advancedrefractory solid tumors.

AquaporinsIon movement is accompanied by water movement. Transcellularwater transport is mediated by aquaporins (AQPs). The maindriving force of AQPs is the osmotic difference between the intra-and the extracellular space. In addition to water transport, AQPsare involved in the transport of smaller molecules or function asion channels. Several mechanisms have been described throughwhich AQPs enhance tumor cell migration and invasion (De Iesoand Yool, 2018). AQP1 facilitates hypoxia-induced angiogenesis,AQP1, -3, -4, -5, and -9 are involved in epithelial-mesenchymaltransition and extracellular matrix degradation, whereas AQP1-4 enhance cell adhesion (De Ieso and Yool, 2018). Of the13 isoforms currently identified, AQP1, -3, -5, and -8 areassociated with OC. AQP1 is widely expressed in many tissuesand is a potential biomarker in breast cancer (Qin et al.,2016). Yamazato et al. (2018) found a correlation betweenAQP1 expression and the outcome of ESCC patients (Yamazatoet al., 2018). The authors showed that in ESCC patients withhigh AQP1 expression in the cytoplasm, the 5-year survivalrate is lower compared to ESCC patients in whom AQP1 islocalized to the nuclear membrane. AQP1 knock-down in ESCCcell lines inhibits cell proliferation and migration and inducesapoptosis. In addition, microarray analysis shows an interactionbetween AQP1 and death-receptor signaling pathway-relatedgenes, supporting the importance of AQP1 in tumorigenesis.Similar results were observed for AQP3 (Kusayama et al., 2011;Niu et al., 2012). AQP3 is highly expressed in ESCC, andthe absence or decreased function of AQP3 decreases adhesionof cancerous cells via inhibition of integrins and the focaladhesion kinase–mitogen-activated protein kinase (FAK-MAPK)signaling pathway. Inhibition of cell adhesion is associated withdecreased cell growth and apoptosis, indicating the critical roleof AQP3 in cell survival. Increased expression of AQP3 inESCC and EAC has also been demonstrated by Niu et al.(2012). Studies have also reported the importance of AQP5 inESCC (Shimizu et al., 2014). AQP5 down-regulation inhibitscell cycle progression in ESCC cell lines, and microarrayanalysis shows that a lack of AQP5 affects the expressionof genes involved in cellular growth. Studies have shown acorrelation between the size and histological type of ESCCand AQP5 expression in human tumor tissues. In addition,the 3-year progression-free survival is lower in ESCC patientswith AQP5 positivity, which raises the possibility of usingAQP5 as a biomarker. AQP8 has also been implicated in theprogression of OC (Chang et al., 2014). Using human ESCCcell line, Eca-109, it has been shown that the epidermal growthfactor (EGF)-induced cell migration is associated with increasedexpression of AQP8 through the EGF receptor-ERK1/2 signaltransduction pathway.

Inhibitors and Therapeutic PerspectivesSeveral inhibitors have been identified that are more or lesseffective in inhibiting AQPs, such as the small moleculeinhibitors, TEA and acetazolamide, which can selectively andreversibly inhibit AQP1 and -4 isoforms (Abir-Awan et al., 2019).The recently discovered molecules, DFP00173 and Z433927330that have been shown to block glyceroporins by reducing channelglycerol permeability (Abir-Awan et al., 2019). Among the heavymetal ions, Cu ions, NiCl2 and gold compounds proved to beeffective in inhibiting AQP3 (Aikman et al., 2018). HgCl2 isalso widely used to inhibit AQPs, however, the disadvantage ofthis molecule is that it blocks all AQP isoforms and mercuryis highly toxic to cells due to its many side effects (Aikmanet al., 2018). Furthermore, monoclonal antibodies such as anti-AQP4 have been shown to be promising agents in the treatmentof neuromyelitis optica (NMO) (Tradtrantip et al., 2012) andclinical trials are in progress to evaluate the efficacy of anti-AQP4 IgG in NMO and multiple sclerosis. To the best of ourknowledge, no controlled clinical trial has been conducted todate in which the effects of AQP inhibitors have been tested inoesophageal diseases.

CONCLUSION

This review summarized the expression and role of iontransporters in OECs (Tables 1, 2) and described theirpathological function (Supplementary Table S1). Ion transportfundamentally determines both the extra- and the intracellularpH, which affects several intracellular mechanisms, includingthe release of inflammatory mediators or the expression ofcell cycle regulatory proteins. Research increasingly highlightsthe significance of ion transporters in various inflammatoryand cancerous processes, and many of them have emerged aspromising therapeutic targets or independent prognostic factorsin cancer. This review described numerous ion transporterswhose inhibition reduces proliferation and induces apoptosisof cancerous oesophageal cells (Supplementary Table S1).These findings raise the possibility that pharmacologicalinhibition of these ion transporters or a combination ofion transporter inhibitors with chemotherapeutic agents mightprovide alternative treatment options for OC patients who do notrespond to standard treatment.

AUTHOR CONTRIBUTIONS

VV, EB, and MK contributed to conception and design of thestudy. EB and PH wrote separate sections of the manuscript. Allauthors read and approved the submitted version.

FUNDING

This study was supported by the National Research,Development and Innovation Office (FK123982), the

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Economic Development and Innovation OperationalProgramme Grant (GINOP-2.3.2-15-2016-00015), and theNational Research, Development and Innovation Office,by the Ministry of Human Capacities (EFOP 3.6.2-16-2017-00006).

SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be foundonline at: https://www.frontiersin.org/articles/10.3389/fphys.2020.00855/full#supplementary-material

REFERENCESAbdulnour-Nakhoul, S., Bor, S., Imeryuz, N., and Orlando, R. C. (1999).

Mechanisms of basolateral Na+ transport in rabbit esophageal epithelial cells.Am. J. Physiol. 276, G507–G517.

Abdulnour-Nakhoul, S., Nakhoul, H. N., Kalliny, M. I., Gyftopoulos, A., Rabon,E., Doetjes, R., et al. (2011). Ion transport mechanisms linked to bicarbonatesecretion in the esophageal submucosal glands. Am. J. Physiol. Regul. Integr.Comp. Physiol. 301, R83–R96.

Abdulnour-Nakhoul, S., Nakhoul, N. L., Caymaz-Bor, C., and Orlando, R. C.(2002). Chloride transport in rabbit esophageal epithelial cells. Am. J. Physiol.Gastrointest. Liver Physiol. 282, G663–G675.

Abdulnour-Nakhoul, S., Nakhoul, N. L., Wheeler, S. A., Wang, P., Swenson, E. R.,and Orlando, R. C. (2005). HCO3- secretion in the esophageal submucosalglands. Am. J. Physiol. Gastrointest. Liver Physiol. 288, G736–G744.

Abir-Awan, M., Kitchen, P., Salman, M. M., Conner, M. T., Conner, A. C., and Bill,R. M. (2019). Inhibitors of mammalian aquaporin water channels. Int. J. Mol.Sci. 20:1589. doi: 10.3390/ijms20071589

Aikman, B., De Almeida, A., Meier-Menches, S. M., and Casini, A. (2018).Aquaporins in cancer development: opportunities for bioinorganic chemistryto contribute novel chemical probes and therapeutic agents. Metallomics 10,696–712. doi: 10.1039/c8mt00072g

Aires, V., Hichami, A., Boulay, G., and Khan, N. A. (2007). Activation of TRPC6calcium channels by diacylglycerol (DAG)-containing arachidonic acid: acomparative study with DAG-containing docosahexaenoic acid. Biochimie 89,926–937. doi: 10.1016/j.biochi.2006.10.016

Akar, F., Jiang, G., Paul, R. J., and O’neill, W. C. (2001). Contractile regulation of theNa(+)-K(+)-2Cl(-) cotransporter in vascular smooth muscle. Am. J. Physiol.Cell Physiol. 281, C579–C584.

Alper, S. L., Chernova, M. N., Williams, J., Zasloff, M., Law, F. Y., and Knauf, P. A.(1998). Differential inhibition of AE1 and AE2 anion exchangers by oxonoldyes and by novel polyaminosterol analogs of the shark antibiotic squalamine.Biochem. Cell Biol. 76, 799–806. doi: 10.1139/o98-082

Ariyoshi, Y., Shiozaki, A., Ichikawa, D., Shimizu, H., Kosuga, T., Konishi, H., et al.(2017). Na+/H+ exchanger 1 has tumor suppressive activity and prognosticvalue in esophageal squamous cell carcinoma. Oncotarget 8, 2209–2223. doi:10.18632/oncotarget.13645

Awayda, M. S., Bengrine, A., Tobey, N. A., Stockand, J. D., and Orlando, R. C.(2004). Nonselective cation transport in native esophageal epithelia. Am. J.Physiol. Cell Physiol. 287, C395–C402.

Ben-Ari, Y. (2017). NKCC1 chloride importer antagonists attenuate manyneurological and psychiatric disorders. Trends Neurosci. 40, 536–554. doi:10.1016/j.tins.2017.07.001

Ben-Ari, Y., Gaiarsa, J. L., Tyzio, R., and Khazipov, R. (2007). GABA: a pioneertransmitter that excites immature neurons and generates primitive oscillations.Physiol. Rev. 87, 1215–1284. doi: 10.1152/physrev.00017.2006

Blanchard, C., Stucke, E. M., Burwinkel, K., Caldwell, J. M., Collins, M. H.,Ahrens, A., et al. (2010). Coordinate interaction between IL-13 and epithelialdifferentiation cluster genes in eosinophilic esophagitis. J. Immunol. 184, 4033–4041. doi: 10.4049/jimmunol.0903069

Bonar, P. T., and Casey, J. R. (2008). Plasma membrane Cl(-)/HCO(3)(-)exchangers: structure, mechanism and physiology. Channels 2, 337–345. doi:10.4161/chan.2.5.6899

Bouron, A., Chauvet, S., Dryer, S., and Rosado, J. A. (2016). Second messenger-operated calcium entry through TRPC6. Adv. Exp. Med. Biol. 898, 201–249.doi: 10.1007/978-3-319-26974-0_10

Castrop, H., and Schiessl, I. M. (2014). Physiology and pathophysiology of therenal Na-K-2Cl cotransporter (NKCC2). Am. J. Physiol. Renal Physiol. 307,F991–F1002.

Chaitman, B. R. (2003). A review of the GUARDIAN trial results: clinicalimplications and the significance of elevated perioperative CK-MB on 6-monthsurvival. J. Card. Surg. 18(Suppl. 1), 13–20. doi: 10.1046/j.1540-8191.18.s1.3.x

Chang, H., Shi, Y. H., Talaf, T. K., and Lin, C. (2014). Aquaporin-8 mediates humanesophageal cancer Eca-109 cell migration via the EGFR-Erk1/2 pathway. Int. J.Clin. Exp. Pathol. 7, 7663–7671.

Choi, S., Cui, C., Luo, Y., Kim, S. H., Ko, J. K., Huo, X., et al. (2018). Selectiveinhibitory effects of zinc on cell proliferation in esophageal squamous cellcarcinoma through Orai1. FASEB J. 32, 404–416. doi: 10.1096/fj.201700227rrr

Collins, M. H. (2008). Histopathologic features of eosinophilic esophagitis.Gastrointest. Endosc. Clin. N. Am. 18, 59–71. doi: 10.1016/j.giec.2007.09.014

Cotton, C. U. (2000). Basolateral potassium channels and epithelial ion transport.Am. J. Respir. Cell Mol. Biol. 23, 270–272. doi: 10.1165/ajrcmb.23.3.f198

Cui, C., Chang, Y., Zhang, X., Choi, S., Tran, H., Penmetsa, K. V., et al. (2018).Targeting Orai1-mediated store-operated calcium entry by RP4010 for anti-tumor activity in esophagus squamous cell carcinoma. Cancer Lett. 432, 169–179. doi: 10.1016/j.canlet.2018.06.006

Czepan, M., Rakonczay, Z., Varro, A., Steele, I., Dimaline, R., Lertkowit, N.,et al. (2012). NHE1 activity contributes to migration and is necessary forproliferation of human gastric myofibroblasts. Pflugers Arch. 463, 459–475.doi: 10.1007/s00424-011-1059-6

De Ieso, M. L., and Yool, A. J. (2018). Mechanisms of aquaporin-facilitated cancerinvasion and metastasis. Front. Chem. 6:135. doi: 10.3389/fchem.2018.00135

Delpire, E., and Lauf, P. K. (1992). Kinetics of DIDS inhibition of swelling-activatedK-Cl cotransport in low K sheep erythrocytes. J. Membr. Biol. 126, 89–96.

Delpire, E., and Weaver, C. D. (2016). Challenges of finding novel drugs targetingthe K-Cl cotransporter. ACS Chem. Neurosci. 7, 1624–1627. doi: 10.1021/acschemneuro.6b00366

Demaurex, N., and Grinstein, S. (1994). Na+/H+ antiport: modulation by ATPand role in cell volume regulation. J. Exp. Biol. 196, 389–404.

Denker, S. P., Huang, D. C., Orlowski, J., Furthmayr, H., and Barber, D. L. (2000).Direct binding of the Na–H exchanger NHE1 to ERM proteins regulates thecortical cytoskeleton and cell shape independently of H(+) translocation. Mol.Cell 6, 1425–1436. doi: 10.1016/s1097-2765(00)00139-8

Ding, X., He, Z., Shi, Y., Wang, Q., and Wang, Y. (2010). Targeting TRPC6 channelsin oesophageal carcinoma growth. Expert Opin. Ther. Targets 14, 513–527.doi: 10.1517/14728221003733602

Ding, X. W., Luo, H. S., Luo, B., Xu, D. Q., and Gao, S. (2008a). Overexpressionof hERG1 in resected esophageal squamous cell carcinomas: a marker for poorprognosis. J. Surg. Oncol. 97, 57–62. doi: 10.1002/jso.20891

Ding, X. W., Wang, X. G., Luo, H. S., Tan, S. Y., Gao, S., Luo, B., et al. (2008b).Expression and prognostic roles of Eag1 in resected esophageal squamous cellcarcinomas. Dig. Dis. Sci. 53, 2039–2044. doi: 10.1007/s10620-007-0116-7

Djamgoz, M. B., Coombes, R. C., and Schwab, A. (2014). Ion transport andcancer: from initiation to metastasis. Philos. Trans. R. Soc. Lond. B Biol. Sci.369:20130092. doi: 10.1098/rstb.2013.0092

Donowitz, M., Ming Tse, C., and Fuster, D. (2013). SLC9/NHE gene family, aplasma membrane and organellar family of Na(+)/H(+) exchangers. Mol.Aspects Med. 34, 236–251. doi: 10.1016/j.mam.2012.05.001

Duvvuri, U., Shiwarski, D. J., Xiao, D., Bertrand, C., Huang, X., Edinger, R. S., et al.(2012). TMEM16A induces MAPK and contributes directly to tumorigenesisand cancer progression. Cancer Res. 72, 3270–3281. doi: 10.1158/0008-5472.can-12-0475-t

Enzinger, P. C., and Mayer, R. J. (2003). Esophageal cancer. N. Engl. J. Med. 349,2241–2252.

Farkas, K., Yeruva, S., Rakonczay, Z., Ludolph, L., Molnar, T., Nagy, F., et al. (2011).New therapeutic targets in ulcerative colitis: the importance of ion transportersin the human colon. Inflamm. Bowel Dis. 17, 884–898. doi: 10.1002/ibd.21432

Frontiers in Physiology | www.frontiersin.org 12 July 2020 | Volume 11 | Article 855

fphys-11-00855 July 16, 2020 Time: 11:16 # 13

Becskeházi et al. Ion Transport in the Osophagus

Fitzgerald, R. C., Omary, M. B., and Triadafilopoulos, G. (1998). Altered sodium-hydrogen exchange activity is a mechanism for acid-induced hyperproliferationin Barrett’s esophagus. Am. J. Physiol. 275, G47–G55.

Frelin, C., Vigne, P., Barbry, P., and Lazdunski, M. (1986). Interaction ofguanidinium and guanidinium derivatives with the Na+/H+ exchange system.Eur. J. Biochem. 154, 241–245. doi: 10.1111/j.1432-1033.1986.tb09388.x

Frizzell, R. A., and Hanrahan, J. W. (2012). Physiology of epithelial chloride andfluid secretion. Cold Spring Harb. Perspect. Med. 2:a009563. doi: 10.1101/cshperspect.a009563

Fujiwara, Y., Higuchi, K., Tominaga, K., Watanabe, T., Oshitani, N., andArakawa, T. (2005). Functional oesophageal epithelial defense against acid.Inflammopharmacology 13, 1–13. doi: 10.1163/156856005774423953

Furuta, G. T., and Katzka, D. A. (2015). Eosinophilic Esophagitis. N. Engl. J. Med.373, 1640–1648.

Garcia-Quiroz, J., Gonzalez-Gonzalez, M. E., Diaz, L., Ordaz-Rosado, D., Segovia-Mendoza, M., Prado-Garcia, H., et al. (2019). Astemizole, an inhibitor ofEther-a-Go-Go-1 potassium channel, increases the activity of the tyrosinekinase inhibitor Gefitinib in breast cancer cells. Rev. Invest. Clin. 71, 186–194.

Goldman, A., Chen, H., Khan, M. R., Roesly, H., Hill, K. A., Shahidullah, M., et al.(2011). The Na+/H+ exchanger controls deoxycholic acid-induced apoptosisby a H+-activated, Na+-dependent ionic shift in esophageal cells. PLoS One6:e23835. doi: 10.1371/journal.pone.0023835

Goldman, A., Shahidullah, M., Goldman, D., Khailova, L., Watts, G., Delamere, N.,et al. (2010). A novel mechanism of acid and bile acid-induced DNA damageinvolving Na+/H+ exchanger: implication for Barrett’s oesophagus. Gut 59,1606–1616. doi: 10.1136/gut.2010.213686

Goldstein, J. L., Fogelson, B. G., Snow, J. C., Schmidt, L. N., Mozwecz, H., andLayden, T. J. (1993). Rabbit esophageal cells possess K+ channels: effect ofhyposmotic stress on channel activity. Gastroenterology 104, 417–426. doi:10.1016/0016-5085(93)90409-6

Goldstein, J. L., Watkins, J. L., Greager, J. A., and Layden, T. J. (1994).The esophageal mucosal resistance: structure and function of an uniquegastrointestinal epithelial barrier. J. Lab. Clin. Med. 123, 653–659.

Grinstein, S., Rotin, D., and Mason, M. J. (1989). Na+/H+ exchange and growthfactor-induced cytosolic pH changes. Role in cellular proliferation. Biochim.Biophys. Acta 988, 73–97. doi: 10.1016/0304-4157(89)90004-x

Guan, B., Hoque, A., and Xu, X. (2014). Amiloride and guggulsterone suppressionof esophageal cancer cell growth in vitro and in nude mouse xenografts. Front.Biol. 9, 75–81. doi: 10.1007/s11515-014-1289-z

Guarino, M. P., Cheng, L., Ma, J., Harnett, K., Biancani, P., Altomare, A., et al.(2010). Increased TRPV1 gene expression in esophageal mucosa of patientswith non-erosive and erosive reflux disease. Neurogastroenterol. Motil. 22,746–51.e219.

Gunther, C., Neumann, H., and Vieth, M. (2014). Esophageal epithelial resistance.Dig. Dis. 32, 6–10. doi: 10.1159/000357001

Haas, M., and Forbush, B. (1998). The Na-K-Cl cotransporters. J. Bioenerg.Biomembr. 30, 161–172.

Haas, M., and Forbush, B. (2000). The Na-K-Cl cotransporter of secretory epithelia.Annu. Rev. Physiol. 62, 515–534. doi: 10.1146/annurev.physiol.62.1.515

Hampel, P., Romermann, K., Macaulay, N., and Loscher, W. (2018). Azosemideis more potent than bumetanide and various other loop diuretics to inhibitthe sodium-potassium-chloride-cotransporter human variants hNKCC1A andhNKCC1B. Sci. Rep. 8:9877.

Hegyi, P., Pandol, S., Venglovecz, V., and Rakonczay, Z. Jr. (2011). The acinar-ductal tango in the pathogenesis of acute pancreatitis. Gut 60, 544–552. doi:10.1136/gut.2010.218461

Heitzmann, D., and Warth, R. (2008). Physiology and pathophysiology ofpotassium channels in gastrointestinal epithelia. Physiol. Rev. 88, 1119–1182.doi: 10.1152/physrev.00020.2007

Hofmann, T., Schaefer, M., Schultz, G., and Gudermann, T. (2000). Transientreceptor potential channels as molecular substrates of receptor-mediated cationentry. J. Mol. Med. 78, 14–25. doi: 10.1007/s001090050378

Holzer, P. (2008). The pharmacological challenge to tame the transient receptorpotential vanilloid-1 (TRPV1) nocisensor. Br. J. Pharmacol. 155, 1145–1162.doi: 10.1038/bjp.2008.351

Huang, F., Zhang, H., Wu, M., Yang, H., Kudo, M., Peters, C. J., et al. (2012).Calcium-activated chloride channel TMEM16A modulates mucin secretion

and airway smooth muscle contraction. Proc. Natl. Acad. Sci. U.S.A. 109,16354–16359. doi: 10.1073/pnas.1214596109

Huang, R., Wang, F., Yang, Y., Ma, W., Lin, Z., Cheng, N., et al. (2019). Recurrentactivations of transient receptor potential vanilloid-1 and vanilloid-4 promotecellular proliferation and migration in esophageal squamous cell carcinomacells. FEBS Open Bio 9, 206–225. doi: 10.1002/2211-5463.12570

Humphreys, B. D., Jiang, L., Chernova, M. N., and Alper, S. L. (1994). Functionalcharacterization and regulation by pH of murine AE2 anion exchangerexpressed in Xenopus oocytes. Am. J. Physiol. 267, C1295–C1307.

Janne, P. A., Yang, J. C., Kim, D. W., Planchard, D., Ohe, Y., Ramalingam, S. S.,et al. (2015). AZD9291 in EGFR inhibitor-resistant non-small-cell lung cancer.N. Engl. J. Med. 372, 1689–1699.

Judak, L., Hegyi, P., Rakonczay, Z., Maleth, J., Gray, M. A., and Venglovecz,V. (2014). Ethanol and its non-oxidative metabolites profoundly inhibitCFTR function in pancreatic epithelial cells which is prevented by ATPsupplementation. Pflugers Arch. 466, 549–562. doi: 10.1007/s00424-013-1333-x

Jung, J., Nam, J. H., Park, H. W., Oh, U., Yoon, J. H., and Lee, M. G.(2013). Dynamic modulation of ANO1/TMEM16A HCO3(-) permeability byCa2+/calmodulin. Proc. Natl. Acad. Sci. U.S.A. 110, 360–365. doi: 10.1073/pnas.1211594110

Kere, J. (2006). Overview of the SLC26 family and associated diseases. NovartisFound. Symp. 273, 2–11.

Khalbuss, W. E., Alkiek, R., Marousis, C. G., and Orlando, R. C. (1993). Potassiumconductance in rabbit esophageal epithelium. Am. J. Physiol. 265, G28–G34.

Khan, H. Y., Mpilla, G. B., Sexton, R., Viswanadha, S., Penmetsa, K. V.,Aboukameel, A., et al. (2020). Calcium Release-Activated Calcium(CRAC) channel inhibition suppresses pancreatic ductal adenocarcinomacell proliferation and patient-derived tumor growth. Cancers 12:750.doi: 10.3390/cancers12030750

Kleyman, T. R., and Cragoe, E. J. (1988). Amiloride and its analogs as tools in thestudy of ion transport. J. Membr. Biol. 105, 1–21. doi: 10.1007/bf01871102

Klingelhofer, D., Zhu, Y., Braun, M., Bruggmann, D., Schoffel, N., and Groneberg,D. A. (2019). A world map of esophagus cancer research: a critical accounting.J. Transl. Med. 17:150.

Kusayama, M., Wada, K., Nagata, M., Ishimoto, S., Takahashi, H., Yoneda, M.,et al. (2011). Critical role of aquaporin 3 on growth of human esophageal andoral squamous cell carcinoma. Cancer Sci. 102, 1128–1136. doi: 10.1111/j.1349-7006.2011.01927.x

Lacroix, J., Poet, M., Maehrel, C., and Counillon, L. (2004). A mechanism forthe activation of the Na/H exchanger NHE-1 by cytoplasmic acidification andmitogens. EMBO Rep. 5, 91–96. doi: 10.1038/sj.embor.7400035

Laczko, D., Rosztoczy, A., Birkas, K., Katona, M., Rakonczay, Z., Tiszlavicz, L., et al.(2016). Role of ion transporters in the bile acid-induced esophageal injury. Am.J. Physiol. Gastrointest. Liver Physiol. 311, G16–G31.

Lastraioli, E., Lottini, T., Iorio, J., Freschi, G., Fazi, M., Duranti, C., et al. (2016).hERG1 behaves as biomarker of progression to adenocarcinoma in Barrett’sesophagus and can be exploited for a novel endoscopic surveillance. Oncotarget7, 59535–59547. doi: 10.18632/oncotarget.11149

Lastraioli, E., Taddei, A., Messerini, L., Comin, C. E., Festini, M., Giannelli, M.,et al. (2006). hERG1 channels in human esophagus: evidence for their aberrantexpression in the malignant progression of Barrett’s esophagus. J. Cell. Physiol.209, 398–404. doi: 10.1002/jcp.20748

Layden, T. J., Agnone, L. M., Schmidt, L. N., Hakim, B., and Goldstein, J. L.(1990). Rabbit esophageal cells possess an Na+,H+ antiport. Gastroenterology99, 909–917. doi: 10.1016/0016-5085(90)90606-2

Layden, T. J., Schmidt, L., Agnone, L., Lisitza, P., Brewer, J., and Goldstein, J. L.(1992). Rabbit esophageal cell cytoplasmic pH regulation: role of Na+-H+antiport and H+-dependent HCO3- transport systems. Am. J. Physiol. 263,G407–G413.

Lee, M. G., Ohana, E., Park, H. W., Yang, D., and Muallem, S. (2012). Molecularmechanism of pancreatic and salivary gland fluid and HCO3 secretion. Physiol.Rev. 92, 39–74. doi: 10.1152/physrev.00011.2011

Lee, S., Cil, O., Haggie, P. M., and Verkman, A. S. (2019). 4,8-DimethylcoumarinInhibitors of Intestinal Anion Exchanger slc26a3 (Downregulated in Adenoma)for Anti-Absorptive Therapy of Constipation. J. Med. Chem. 62, 8330–8337.doi: 10.1021/acs.jmedchem.9b01192

Frontiers in Physiology | www.frontiersin.org 13 July 2020 | Volume 11 | Article 855

fphys-11-00855 July 16, 2020 Time: 11:16 # 14

Becskeházi et al. Ion Transport in the Osophagus

Leuner, K., Kazanski, V., Muller, M., Essin, K., Henke, B., Gollasch, M., et al. (2007).Hyperforin–a key constituent of St. John’s wort specifically activates TRPC6channels. FASEB J. 21, 4101–4111. doi: 10.1096/fj.07-8110com

Li, Y., Zhu, C. L., Nie, C. J., Li, J. C., Zeng, T. T., Zhou, J., et al. (2013). Investigationof tumor suppressing function of CACNA2D3 in esophageal squamous cellcarcinoma. PLoS One 8:e60027. doi: 10.1371/journal.pone.0060027

Lin, B. R., Hsieh, H. T., Lee, J. M., Lai, I. R., Chen, C. F., and Yu, L. C. (2008).Luminal hydrochloric acid stimulates rapid transepithelial ion fluxes in rodentesophageal stratified squamous epithelium. J. Physiol. Pharmacol. 59, 525–542.

Lipskaia, L., and Lompre, A. M. (2004). Alteration in temporal kinetics of Ca2+signaling and control of growth and proliferation. Biol. Cell 96, 55–68. doi:10.1016/j.biolcel.2003.11.001

Liu, Z., Zhang, S., Hou, F., Zhang, C., Gao, J., and Wang, K. (2019). Inhibition ofCa(2+) -activated chloride channel ANO1 suppresses ovarian cancer throughinactivating PI3K/Akt signaling. Int. J. Cancer 144, 2215–2226. doi: 10.1002/ijc.31887

Martial, S. (2016). Involvement of ion channels and transporters in carcinomaangiogenesis and metastasis. Am. J. Physiol. Cell Physiol. 310, C710–C727.

Mentzer, R. M., Bartels, C., Bolli, R., Boyce, S., Buckberg, G. D., Chaitman, B.,et al. (2008). Sodium-hydrogen exchange inhibition by cariporide to reduce therisk of ischemic cardiac events in patients undergoing coronary artery bypassgrafting: results of the EXPEDITION study. Ann. Thorac. Surg. 85, 1261–1270.doi: 10.1016/j.athoracsur.2007.10.054

Mihara, H., Boudaka, A., Sugiyama, T., Moriyama, Y., and Tominaga, M. (2011).Transient receptor potential vanilloid 4 (TRPV4)-dependent calcium influx andATP release in mouse oesophageal keratinocytes. J. Physiol. 589, 3471–3482.doi: 10.1113/jphysiol.2011.207829

Miraglia, E., Viarisio, D., Riganti, C., Costamagna, C., Ghigo, D., and Bosia,A. (2005). Na+/H+ exchanger activity is increased in doxorubicin-resistanthuman colon cancer cells and its modulation modifies the sensitivity of the cellsto doxorubicin. Int. J. Cancer 115, 924–929. doi: 10.1002/ijc.20959

Namkung, W., Phuan, P. W., and Verkman, A. S. (2011). TMEM16A inhibitorsreveal TMEM16A as a minor component of calcium-activated chloride channelconductance in airway and intestinal epithelial cells. J. Biol. Chem. 286, 2365–2374. doi: 10.1074/jbc.m110.175109

Nie, C., Qin, X., Li, X., Tian, B., Zhao, Y., Jin, Y., et al. (2019). CACNA2D3 enhancesthe chemosensitivity of esophageal squamous cell carcinoma to cisplatin viainducing Ca(2+)-mediated apoptosis and suppressing PI3K/Akt pathways.Front. Oncol. 9:185. doi: 10.3389/fonc.2019.00185

Nilius, B., and Owsianik, G. (2011). The transient receptor potential family of ionchannels. Genome Biol. 12:218. doi: 10.1186/gb-2011-12-3-218

Niu, D., Kondo, T., Nakazawa, T., Yamane, T., Mochizuki, K., Kawasaki, T., et al.(2012). Expression of aquaporin3 in human neoplastic tissues. Histopathology61, 543–551. doi: 10.1111/j.1365-2559.2011.04165.x

Oosterwijk, E., and Gillies, R. J. (2014). Targeting ion transport in cancer. Philos.Trans. R. Soc. Lond. B Biol. Sci. 369:20130107. doi: 10.1098/rstb.2013.0107

Orlando, G. S., Tobey, N. A., Wang, P., Abdulnour-Nakhoul, S., and Orlando, R. C.(2002). Regulatory volume decrease in human esophageal epithelial cells. Am.J. Physiol. Gastrointest. Liver Physiol. 283, G932–G937.

Orlando, R. C. (1986). Esophageal epithelial resistance. J. Clin.Gastroenterol. 8(Suppl. 1), 12–16. doi: 10.1097/00004836-198606001-00004

Orlando, R. C., Bryson, J. C., and Powell, D. W. (1984). Mechanisms of H+ injuryin rabbit esophageal epithelium. Am. J. Physiol. 246, G718–G724.

Orlando, R. C., Powell, D. W., and Carney, C. N. (1981). Pathophysiology ofacute acid injury in rabbit esophageal epithelium. J. Clin. Invest. 68, 286–293.doi: 10.1172/jci110246

Park, S. Y., Lee, Y. J., Cho, E. J., Shin, C. Y., and Sohn, U. D. (2015). Intrinsicresistance triggered under acid loading within normal esophageal epithelialcells: NHE1- and ROS-mediated survival. J. Cell. Physiol. 230, 1503–1514. doi:10.1002/jcp.24896

Pedersen, S. F., and Stock, C. (2013). Ion channels and transporters in cancer:pathophysiology, regulation, and clinical potential. Cancer Res. 73, 1658–1661.doi: 10.1158/0008-5472.can-12-4188

Persi, E., Duran-Frigola, M., Damaghi, M., Roush, W. R., Aloy, P., Cleveland,J. L., et al. (2018). Systems analysis of intracellular pH vulnerabilities for cancertherapy. Nat. Commun. 9:2997.

Powell, D. W., Morris, S. M., and Boyd, D. D. (1975). Water and electrolytetransport by rabbit esophagus. Am. J. Physiol. 229, 438–443. doi: 10.1152/ajplegacy.1975.229.2.438

Qin, F., Zhang, H., Shao, Y., Liu, X., Yang, L., Huang, Y., et al. (2016). Expression ofaquaporin1, a water channel protein, in cytoplasm is negatively correlated withprognosis of breast cancer patients. Oncotarget 7, 8143–8154. doi: 10.18632/oncotarget.6994

Qin, N., Yagel, S., Momplaisir, M. L., Codd, E. E., and D’andrea, M. R. (2002).Molecular cloning and characterization of the human voltage-gated calciumchannel alpha(2)delta-4 subunit. Mol. Pharmacol. 62, 485–496. doi: 10.1124/mol.62.3.485

Rees, D. C., Thein, S. L., Osei, A., Drasar, E., Tewari, S., Hannemann, A.,et al. (2015). The clinical significance of K-Cl cotransport activity in red cellsof patients with HbSC disease. Haematologica 100, 595–600. doi: 10.3324/haematol.2014.120402

Rocha-Gonzalez, H. I., Mao, S., and Alvarez-Leefmans, F. J. (2008). Na+,K+,2Cl-cotransport and intracellular chloride regulation in rat primary sensoryneurons: thermodynamic and kinetic aspects. J. Neurophysiol. 100, 169–184.doi: 10.1152/jn.01007.2007

Rolver, M. G., Elingaard-Larsen, L. O., Andersen, A. P., Counillon, L., andPedersen, S. F. (2020). Pyrazine ring-based Na(+)/H(+) exchanger (NHE)inhibitors potently inhibit cancer cell growth in 3D culture, independent ofNHE1. Sci. Rep. 10:5800.

Russell, J. M. (2000). Sodium-potassium-chloride cotransport. Physiol. Rev. 80,211–276. doi: 10.1152/physrev.2000.80.1.211

Santoni, G., and Farfariello, V. (2011). TRP channels and cancer: new targets fordiagnosis and chemotherapy. Endocr. Metab. Immune Disord. Drug Targets 11,54–67. doi: 10.2174/187153011794982068

Seo, Y., Lee, H. K., Park, J., Jeon, D. K., Jo, S., Jo, M., et al. (2016). Ani9, a novelpotent small-molecule ANO1 inhibitor with negligible effect on ANO2. PLoSOne 11:e0155771. doi: 10.1371/journal.pone.0155771

Seo, Y., Ryu, K., Park, J., Jeon, D. K., Jo, S., Lee, H. K., et al. (2017). Inhibitionof ANO1 by luteolin and its cytotoxicity in human prostate cancer PC-3 cells.PLoS One 12:e0174935. doi: 10.1371/journal.pone.0174935

Shallat, S., Schmidt, L., Reaka, A., Rao, D., Chang, E. B., Rao, M. C., et al.(1995). NHE-1 isoform of the Na+/H+ antiport is expressed in the ratand rabbit esophagus. Gastroenterology 109, 1421–1428. doi: 10.1016/0016-5085(95)90626-6

Shang, L., Hao, J. J., Zhao, X. K., He, J. Z., Shi, Z. Z., Liu, H. J., et al. (2016).ANO1 protein as a potential biomarker for esophageal cancer prognosis andprecancerous lesion development prediction. Oncotarget 7, 24374–24382. doi:10.18632/oncotarget.8223

Shapiro, J., Van Lanschot, J. J. B., Hulshof, M., Van Hagen, P., Van Berge, M. I.,Wijnhoven, B. P. L., et al. (2015). Neoadjuvant chemoradiotherapy plus surgeryversus surgery alone for oesophageal or junctional cancer (CROSS): long-termresults of a randomised controlled trial. Lancet Oncol. 16, 1090–1098.

Shi, J., Mori, E., Mori, Y., Mori, M., Li, J., Ito, Y., et al. (2004). Multiple regulation bycalcium of murine homologues of transient receptor potential proteins TRPC6and TRPC7 expressed in HEK293 cells. J. Physiol. 561, 415–432.

Shi, Y., Ding, X., He, Z. H., Zhou, K. C., Wang, Q., and Wang, Y. Z. (2009). Criticalrole of TRPC6 channels in G2 phase transition and the development of humanoesophageal cancer. Gut 58, 1443–1450. doi: 10.1136/gut.2009.181735

Shikano, M., Ueda, T., Kamiya, T., Ishida, Y., Yamada, T., Mizushima, T., et al.(2011). Acid inhibits TRPV4-mediated Ca(2)(+) influx in mouse esophagealepithelial cells. Neurogastroenterol. Motil. 23, 1020–1028, e1497.

Shimizu, H., Shiozaki, A., Ichikawa, D., Fujiwara, H., Konishi, H., Ishii, H., et al.(2014). The expression and role of Aquaporin 5 in esophageal squamous cellcarcinoma. J. Gastroenterol. 49, 655–666.

Shiozaki, A., Hikami, S., Ichikawa, D., Kosuga, T., Shimizu, H., Kudou, M., et al.(2018). Anion exchanger 2 suppresses cellular movement and has prognosticsignificance in esophageal squamous cell carcinoma. Oncotarget 9, 25993–26006. doi: 10.18632/oncotarget.25417

Shiozaki, A., Kudou, M., Ichikawa, D., Shimizu, H., Arita, T., Kosuga, T., et al.(2017). Expression and role of anion exchanger 1 in esophageal squamous cellcarcinoma. Oncotarget 8, 17921–17935. doi: 10.18632/oncotarget.14900

Shiozaki, A., Nako, Y., Ichikawa, D., Konishi, H., Komatsu, S., Kubota, T.,et al. (2014a). Role of the Na (+)/K (+)/2Cl(-) cotransporter NKCC1 in cell

Frontiers in Physiology | www.frontiersin.org 14 July 2020 | Volume 11 | Article 855

fphys-11-00855 July 16, 2020 Time: 11:16 # 15

Becskeházi et al. Ion Transport in the Osophagus

cycle progression in human esophageal squamous cell carcinoma. World J.Gastroenterol. 20, 6844–6859.

Shiozaki, A., Takemoto, K., Ichikawa, D., Fujiwara, H., Konishi, H., Kosuga, T.,et al. (2014b). The K-Cl cotransporter KCC3 as an independent prognosticfactor in human esophageal squamous cell carcinoma. Biomed Res. Int.2014:936401.

Siddique, I., and Khan, I. (2003). Regulation of Na/H exchanger-1 ingastroesophageal reflux disease: possible interaction of histamine receptor. Dig.Dis. Sci. 48, 1832–1838.

Silva, R. O., Bingana, R. D., Sales, T., Moreira, R. L. R., Costa, D. V. S., Sales,K. M. O., et al. (2018). Role of TRPV1 receptor in inflammation and impairmentof esophageal mucosal integrity in a murine model of nonerosive reflux disease.Neurogastroenterol. Motil. e13340. doi: 10.1111/nmo.13340 [Epub ahead ofprint].

Slepkov, E. R., Rainey, J. K., Sykes, B. D., and Fliegel, L. (2007). Structural andfunctional analysis of the Na+/H+ exchanger. Biochem. J. 401, 623–633. doi:10.1042/bj20061062

Snow, J. C., Goldstein, J. L., Schmidt, L. N., Lisitza, P., and Layden, T. J. (1993).Rabbit esophageal cells show regulatory volume decrease: ionic basis and effectof pH. Gastroenterology 105, 102–110. doi: 10.1016/0016-5085(93)90015-5

Song, Y., Gao, J., Guan, L., Chen, X., Gao, J., and Wang, K. (2018). Inhibitionof ANO1/TMEM16A induces apoptosis in human prostate carcinoma cells byactivating TNF-alpha signaling. Cell Death Dis. 9:703.

Spechler, S. J., and Souza, R. F. (2014). Barrett’s esophagus. N. Engl. J. Med. 371,836–845.

Stock, C., and Schwab, A. (2015). Ion channels and transporters in metastasis.Biochim. Biophys. Acta 1848, 2638–2646. doi: 10.1016/j.bbamem.2014.11.012

Suzuki, N., Mihara, H., Nishizono, H., Tominaga, M., and Sugiyama, T.(2015). Protease-activated receptor-2 Up-Regulates transient receptor potentialvanilloid 4 function in mouse esophageal Keratinocyte. Dig. Dis. Sci. 60, 3570–3578. doi: 10.1007/s10620-015-3822-6

Tobey, N. A., Cragoe, E. J., and Orlando, R. C. (1995). HCl-induced cell edema inrabbit esophageal epithelium: a bumetanide-sensitive process. Gastroenterology109, 414–421. doi: 10.1016/0016-5085(95)90328-3

Tobey, N. A., Koves, G., and Orlando, R. C. (1998). Human esophageal epithelialcells possess an Na+/H+ exchanger for H+ extrusion. Am. J. Gastroenterol. 93,2075–2081. doi: 10.1111/j.1572-0241.1998.00596.x

Tobey, N. A., Reddy, S. P., Khalbuss, W. E., Silvers, S. M., Cragoe, E. J.,and Orlando, R. C. (1993). Na(+)-dependent and -independent Cl-/HCO3-exchangers in cultured rabbit esophageal epithelial cells. Gastroenterology 104,185–195. doi: 10.1016/0016-5085(93)90851-3

Toth-Molnar, E., Venglovecz, V., Ozsvari, B., Rakonczay, Z., Varro, A., Papp, J. G.,et al. (2007). New experimental method to study acid/base transporters andtheir regulation in lacrimal gland ductal epithelia. Invest. Ophthalmol. Vis. Sci.48, 3746–3755.

Tradtrantip, L., Zhang, H., Saadoun, S., Phuan, P. W., Lam, C., Papadopoulos,M. C., et al. (2012). Anti-aquaporin-4 monoclonal antibody blocker therapyfor neuromyelitis optica. Ann. Neurol. 71, 314–322. doi: 10.1002/ana.22657

Ueda, T., Shikano, M., Kamiya, T., Joh, T., and Ugawa, S. (2011). The TRPV4channel is a novel regulator of intracellular Ca2+ in human esophagealepithelial cells. Am. J. Physiol. Gastrointest. Liver Physiol. 301, G138–G147.

Vanoni, S., Zeng, C., Marella, S., Uddin, J., Wu, D., Arora, K., et al. (2020).Identification of anoctamin 1 (ANO1) as a key driver of esophageal epithelialproliferation in eosinophilic esophagitis. J. Allergy Clin. Immunol. 145, 239–254.e2. doi: 10.1016/j.jaci.2019.07.049

Vashisht, A., Trebak, M., and Motiani, R. K. (2015). STIM and Orai proteinsas novel targets for cancer therapy. A review in the theme: cell andmolecular processes in cancer metastasis. Am. J. Physiol. Cell Physiol 309,C457–C469.

Venglovecz, V., Hegyi, P., Rakonczay, Z., Tiszlavicz, L., Nardi, A., Grunnet,M., et al. (2011). Pathophysiological relevance of apical large-conductanceCa(2)+-activated potassium channels in pancreatic duct epithelial cells. Gut 60,361–369. doi: 10.1136/gut.2010.214213

Venglovecz, V., Rakonczay, Z., Ozsvari, B., Takacs, T., Lonovics, J., Varro, A., et al.(2008). Effects of bile acids on pancreatic ductal bicarbonate secretion in guineapig. Gut 57, 1102–1112. doi: 10.1136/gut.2007.134361

Walker, D., and De Waard, M. (1998). Subunit interaction sites in voltage-dependent Ca2+ channels: role in channel function. Trends Neurosci. 21,148–154. doi: 10.1016/s0166-2236(97)01200-9

Wanajo, A., Sasaki, A., Nagasaki, H., Shimada, S., Otsubo, T., Owaki, S., et al.(2008). Methylation of the calcium channel-related gene, CACNA2D3, isfrequent and a poor prognostic factor in gastric cancer. . Gastroenterology 135,580–590.

Wu, W., Dong, M. Q., Wu, X. G., Sun, H. Y., Tse, H. F., Lau, C. P., et al. (2012).Human ether-a-go-go gene potassium channels are regulated by EGFR tyrosinekinase. Biochim. Biophys. Acta 1823, 282–289. doi: 10.1016/j.bbamcr.2011.10.010

Wulamu, W., Yisireyili, M., Aili, A., Takeshita, K., Alimujiang, A., Aipire, A.,et al. (2019). Chronic stress augments esophageal inflammation, and altersthe expression of transient receptor potential vanilloid 1 and proteaseactivatedreceptor 2 in a murine model. Mol. Med. Rep. 19, 5386–5396.

Xue, H., Wang, Y., Maccormack, T. J., Lutes, T., Rice, C., Davey, M., et al. (2018).Inhibition of Transient Receptor Potential Vanilloid 6 channel, elevated inhuman ovarian cancers, reduces tumour growth in a xenograft model. J. Cancer9, 3196–3207. doi: 10.7150/jca.20639

Yamazato, Y., Shiozaki, A., Ichikawa, D., Kosuga, T., Shoda, K., Arita, T.,et al. (2018). Aquaporin 1 suppresses apoptosis and affects prognosis inesophageal squamous cell carcinoma. Oncotarget 9, 29957–29974. doi: 10.18632/oncotarget.25722

Yu, X., Yu, M., Liu, Y., and Yu, S. (2016). TRP channel functions in thegastrointestinal tract. Semin. Immunopathol. 38, 385–396. doi: 10.1007/s00281-015-0528-y

Zeng, C., Vanoni, S., Wu, D., Caldwell, J. M., Wheeler, J. C., Arora, K., et al. (2018).Solute carrier family 9, subfamily A, member 3 (SLC9A3)/sodium-hydrogenexchanger member 3 (NHE3) dysregulation and dilated intercellular spaces inpatients with eosinophilic esophagitis. J. Allergy Clin. Immunol. 142, 1843–1855.doi: 10.1016/j.jaci.2018.03.017

Zhang, D. Y., Wang, Y., Lau, C. P., Tse, H. F., and Li, G. R. (2008). Both EGFRkinase and Src-related tyrosine kinases regulate human ether-a-go-go-relatedgene potassium channels. Cell. Signal. 20, 1815–1821. doi: 10.1016/j.cellsig.2008.06.006

Zhang, S. S., Wen, J., Yang, F., Cai, X. L., Yang, H., Luo, K. J., et al. (2013). Highexpression of transient potential receptor C6 correlated with poor prognosis inpatients with esophageal squamous cell carcinoma. Med. Oncol. 30:607.

Zhang, Y., Zhang, Y., Fan, H., Shi, Y., Zhan, C., and Wang, Q. (2017). Study onthe role of transient receptor potential C6 channels in esophageal squamouscell carcinoma radiosensitivity. J. Thorac. Dis. 9, 3802–3809. doi: 10.21037/jtd.2017.09.108

Zhang, Z., Wu, X., Zhang, L., Mao, A., Ma, X., and He, D. (2020). Menthol relievesacid reflux inflammation by regulating TRPV1 in esophageal epithelial cells.Biochem. Biophys. Res. Commun. 525, 113–120. doi: 10.1016/j.bbrc.2020.02.050

Zhu, H., Zhang, H., Jin, F., Fang, M., Huang, M., Yang, C. S., et al. (2014). ElevatedOrai1 expression mediates tumor-promoting intracellular Ca2+ oscillations inhuman esophageal squamous cell carcinoma. Oncotarget 5, 3455–3471. doi:10.18632/oncotarget.1903

Conflict of Interest: The authors declare that the research was conducted in theabsence of any commercial or financial relationships that could be construed as apotential conflict of interest.

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